The overall goal of this proposal is to develop a non-invasive, real-time, quantifiable cell-based assay to detect and report on furin-like protease activity to identify small molecule inhibitors of furin-like proteases by high throughput screening (HTS). Furin-like proteolytic enzymes are members of the Proprotein Convertase (PC) family that serve to process immature latent proteins, including growth factors and hormones, receptors, plasma proteins, and matrix metalloproteases containing a specific recognition cleavage motif (RX(K/R)R?), to their mature or functional forms. Processing by furin-like protease family members, such as furin, PACE4, PC5/6, and PC7/8, contributes to development of several degenerative diseases, such as Alzheimer's disease, arteriolosclerosis, and arthritis. Furin-like protease expression and activity is necessary for processing substrates that enhance the cancer phenotype, contributing to cell transformation, tumor progression, metastasis, and angiogenesis. Further, furin-like proteolytic processing of viral coat glycoproteins is required for propagation of infectious viruses such as H5N1 avian influenza, HIV-1, human papillomavirus, ebola, yellow fever, and SARS-CoV. Furin-like proteases activate bacterial toxins found in anthrax, shigella, botulinum, pseudomonas, and diphtheria. Inhibition of furin-like proteolytic activity has been shown to halt toxicity of bacterial toxins, infectivity of viruses, and motility of cancer cells. We hypothesize that inhibiting furin-like proteolytic activity may lead to development of a therapeutic drug that inhibits a broad-spectrum of furin-like protease mediated disease. To aid in experimentation of this hypothesis, in specific aim 1A, we will develop a furin-like protease reporter, which non-invasively and quantitatively senses furin-like protease activity in real time and characterize its specificity and sensitivity to furin-like protease activity. In specific aim 1B, we will miniaturize this assay to adapt it to HTS. In specific aim 1C, we will perform HTS of several specialized small molecule libraries containing 71K compounds to identify furin-like protease inhibitory molecules. In specific aim 2A, a secondary screen will be employed to eliminate false positives, cytotoxic, and non-specific inhibitory molecules. Potency will be assessed by exposing the furin-reporter cells to various concentrations of the candidate compound to determine pIC50 values. In specific aim 2B, we subject the five most efficacious compounds to further validation by determining inhibition (IC50 value) of furin processing of physiological substrates using western blot analysis. Additionally, cytotoxicity will be gauged using cell proliferation assays. In specific aim 2C, the compound's ability to inhibit furin will be confirmed using purified furin in vitro. We will also investigate the molecule's specificity by performing in vitro inhibition assays with other serine proteases. At the conclusion of phase I, we expect to have identified at least one compound or derivative with IC50 <1uM that will be the subject of further analysis and targeted for drug development to treat furin-mediated diseases such as anthrax and cancer in subsequent years. PUBLIC HEALTH RELEVANCE: Millions of people worldwide are exposed to and/or contract furin-like protease mediated diseases such as HIV-1, ebola, avian influenza, human papillomavirus, yellow fever, SARS-CoV, anthrax, botulinum, measles, pseudomonas, shigella, diphtheria, arthritis, arteriosclerosis, Alzheimer's disease, and malignant cancer. Instead of searching for a therapeutic to address each pathogen and disease individually, targeting a single cellular protease may allow defeat of a broad spectrum of furin-like protease mediated disease. The studies described here will result in identification of a molecule that inhibits furin-like proteases and thus may be used to treat the diseases listed above.